Separation of blood and lymphatic vessels requires the hematopoietic signaling proteins SYK and SLP-76, but how blood cells perform this function is not known. Using genetic approaches in mice we have recently identified platelets as the cell type required for blood-lymphatic separation. Genetic fate-mapping studies exclude hematopoietic cell contribution to vascular endothelium during this process, while transgenic expression of SLP-76 in GATA1+ lineages including platelets restores blood-lymphatic separation in SLP-76- deficient animals. Conditional deletion of Slp-76 specifically in platelets is sufficient to confer vascular mixing. We find that Podoplanin (PDPN), a cell surface protein expressed specifically by lymphatic endothelium, is required in a common pathway with SLP-76 during vascular separation. PDPN activates platelets and we detect platelet micro-thrombi on lymphatic endothelium during blood flow ex vivo and at blood-lymphatic separation points in the developing mouse embryo. These studies reveal a previously un-appreciated non- hemostatic role for circulating blood platelets in mediating vascular separation and an unexpected mechanism by which lymphatic vessel growth and development is regulated. Platelets are anuclear blood cells that mediate hemostasis following vessel injury and form the thrombi that underlie stroke and heart attacks. How platelets regulate lymphatic endothelial growth and development is unknown and this proposal will address this question using both in vivo and ex vivo approaches. These studies will explore a completely new cellular and molecular pathway that links lymphatic vascular biology to the blood vascular system and hemostasis with important implications for blood and lymphatic vascular human diseases.